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United States Patent |
5,308,750
|
Mehta
,   et al.
|
May 3, 1994
|
Monoclonal antibodies to putative HCV E2/NS1 proteins and methods for
using same
Abstract
Monoclonal antibodies which specifically bind to Hepatitis C Virus (HCV)
E2/NS1 antigen. Also provided are hybridoma cell lines which secrete these
monoclonal antibodies, methods for using these monoclonal antibodies, and
assay kits for assays which contain these monoclonal antibodies.
Inventors:
|
Mehta; Smriti U. (Libertyville, IL);
Johnson; Jill E. (Waukegan, IL);
Dailey; Stephen H. (Vernon Hills, IL);
Desai; Suresh M. (Libertyville, IL);
Devare; Sushil G. (Northbrook, IL)
|
Assignee:
|
Abbott Laboratories (Abbott Park, IL)
|
Appl. No.:
|
748292 |
Filed:
|
August 21, 1991 |
Current U.S. Class: |
435/5; 435/339; 436/548; 436/820; 530/388.3 |
Intern'l Class: |
C12N 005/00; C12Q 001/70 |
Field of Search: |
435/5,70.21,172.2,240.27
530/388.3
436/548,518,820
|
References Cited
U.S. Patent Documents
5106726 | Apr., 1992 | Wang | 435/5.
|
Foreign Patent Documents |
0318216 | May., 1989 | EP.
| |
Primary Examiner: Kepplinger; Esther L.
Assistant Examiner: Wortman; Donna C.
Attorney, Agent or Firm: Porembski; Priscilla E.
Parent Case Text
This application is a continuation-in-part of U.S. patent application Ser.
Nos. 07/456,162 and 07/610,180, both abandoned, entitled HEPATITIS C
ASSAY, which enjoy common ownership and are incorporated herein by
reference.
Claims
We claim:
1. A monoclonal antibody which specifically binds to Hepatitis C Virus
(HCV) E2/NS1 antigen, wherein said monoclonal antibody is the monoclonal
antibody secreted by hybridoma cell line ATCC deposit No. HB 10856.
2. A monoclonal antibody which specifically binds to Hepatitis C Virus
(HCV) E2/NS1 antigen, wherein said monoclonal antibody is the monoclonal
antibody secreted by hybridoma cell line ATCC deposit No. HB 10857.
3. A hybridoma cell line which secretes a monoclonal antibody which
specifically binds to Hepatitis C Virus (HCV) E2/NS1 antigen and wherein
said hybridoma cell line is A.T.C.C. deposit No. HB 10856.
4. A hybridoma cell line which secretes a monoclonal antibody which
specifically binds to Hepatitis C Virus (HCV) E2/NS1 antigen and wherein
said hybridoma cell line is A.T.C.C. deposit No. HB 10857.
5. A method for determining the presence of Hepatitis C Virus (HCV) antigen
in a test sample which may contain HCV, comprising:
a. contacting the test sample with at least one anti-HCV E2/NS1 antibody
attached to a solid phase which antibody specifically binds to HCV E2/NS1
antigen, to form a mixture;
b. incubating said mixture for a time and under conditions sufficient to
form antigen/antibody complexes;
c. contacting said complexes with an indicator reagent comprising a signal
generating compound which generates a measurable detectable signal
attached to an anti-HCV E2/NS1 antibody, to form a second mixture;
d. incubating said second mixture for a time and under conditions
sufficient to form antibody/antigen/antibody complexes; and
e. determining the presence of HCV in the test sample by detecting the
measurable signal generated, wherein the amount of HCV present in the test
sample is proportional to said measurable signal, wherein either the
antibody specific for HCV E2/NS1 antigen of step (a) or of step (c) is a
monoclonal antibody secreted by an A.T.C.C hybridoma cell line selected
from the group consisting of A.T.C.C. deposit No. HB 10856 and A.T.C.C.
deposit No. HB 10857.
6. The method of claim 5 wherein the signal generating compound is selected
from the group consisting of a luminescent compound, a chemiluminescent
compound, an enzyme and a radioactive element.
7. The method of claim 5 wherein the anti-HCV antibody attached to the
solid phase is a polyclonal antibody.
8. The method of claim 5 wherein said anti-HCV E2/NS1 antibody attached to
the solid phase is a monoclonal antibody.
9. The method of claim 5 wherein said indicator reagent comprises a signal
generating compound attached to a polyclonal antibody.
10. The method of claim 5 wherein said indicator reagent comprises a signal
generating compound attached to a monoclonal antibody.
11. A method for determining the presence and amount of Hepatitis C Virus
(HCV) which may be present in a test sample, comprising:
a. contacting a test sample with a polyclonal anti-HCV E2/NS1 antibody
attached to a solid phase and an indicator reagent comprising a monoclonal
antibody which specifically binds to HCV E.sub.2 /NS1 antigen attached to
a signal generating compound which generates a measurable detectable
signal, to form a mixture, wherein said monoclonal antibody is a
monoclonal antibody secreted by an A.T.C.C hybridoma cell line selected
from the group consisting of A.T.C.C. deposit No. HB 10856 and A.T.C.C.
deposit No. HB 10857;
b. incubating said mixture for a time and under conditions sufficient to
form antibody/antigen/antibody complexes; and
c. determining the presence of HCV present in the test sample by detecting
the measurable signal as an indication of the presence of HCV in the test
sample, wherein the amount of HCV present in the test sample is
proportional to the measurable signal generated.
12. An assay kit for determining the presence of HCV antigen in a test
sample, comprising:
a container containing at least one monoclonal antibody which specifically
binds to HCV E2/NS1 antigen, wherein said monoclonal antibody is a
monoclonal antibody secreted by an A.T.C.C hybridoma cell line selected
from the group consisting of A.T.C.C. deposit No. HB 10856 and A.T.C.C.
deposit No. HB 10857.
13. A method for determining the presence and amount of HCV which may be
present in a test sample, comprising:
a. contacting a test sample with a monoclonal anti-HCV E2/NS1 antibody
attached to a solid phase and an indicator reagent comprising a polyclonal
antibody which specifically binds to HCV E2/NS1 attached to a signal
generating compound which generates a measurable detectable signal, to
form a mixture, wherein said monoclonal antibody is a monoclonal antibody
secreted by an A.T.C.C hybridoma cell line selected from the group
consisting of A.T.C.C. deposit No. HB 10856 and A.T.C.C. deposit No. HB
10857;
b. incubating said mixture for a time and under conditions sufficient to
form antibody/antigen/antibody complexes; and
c. determining the presence of HCV present in the test sample by detecting
the measurable signal as an indication of the presence of HCV in the test
sample, wherein the amount of HCV present in the test sample is
proportional to the measurable signal generated.
Description
BACKGROUND OF THE INVENTION
This invention relates generally to antibodies which specifically bind to
hepatitis C virus (HCV), and more specifically, relates to a panel of
novel hybridoma cells lines which secrete monoclonal antibodies which
specifically bind to the putative HCV protein E2/NS1, and methods for
using these monoclonal antibodies.
Descriptions of hepatitis diseases causing jaundice and icterus have been
known to man since antiquity. Viral hepatitis is now known to include a
group of viral agents with distinctive viral organization protein
structure and mode of replication, causing hepatitis with different
degrees of severity of hepatic damage through different routes of
transmission. Acute viral hepatitis is clinically diagnosed by
well-defined patient symptoms including jaundice, hepatic tenderness and
an elevated level of liver transaminases such as aspartate transaminase
and alanine transaminase.
Serological assays currently are employed to further distinguish between
hepatitis-A and hepatitis-B. Non-A non-B Hepatitis (NANBH) is a term first
used in 1975 that described cases of post-transfusion hepatitis not caused
by either hepatitis A virus or hepatitis B virus. Feinstone et al., New
Engl. J. Med. 292: 454-457 (1975). The diagnosis of NANBH has been made
primarily by means of exclusion on the basis of serological analysis for
the presence of hepatitis A and hepatitis B. NANBH is responsible for
about 90% of the cases of post-transfusion hepatitis. Hollinger et al. in
N. R. Rose et al., eds., Manual of Clinical Immunology, American Society
for Microbiology, Washington, D.C., 558-572 (1986).
Attempts to identify the NANBH virus by virtue of genomic similarity to one
of the known hepatitis viruses have failed thus far, suggesting that NANBH
virus has a distinctive genomic organization and structure. Fowler et al.,
J. Med. Virol, 12: 205-213 (1983), and Weiner et al., J. Med. Virol, 21:
239-247 (1987). Progress in developing assays to detect antibodies
specific for NANBH has been hampered by difficulties encountered in
identifying antigens associated with the virus. Wands et al., U.S. Pat.
No. 4,870,076; Wands et al., Proc. Natl. Acad. Sci. 83: 6608-6612 (1986);
Ohori et al., J. Med. Virol. 12: 161-178 (1983); Bradley et al., Proc.
Natl. Acad. Sci. 84: 6277-6281 (1987); Akatsuka et al., J. Med. Virol. 20:
43-56 (1986).
In May of 1988, a collaborative effort of Chiron Corporation with the
Centers for Disease Control resulted in the identification of a putative
NANB agent, hepatitis C virus (HCV). M. Houghton et al. cloned and
expressed in E. coli a NANB agent obtained from the infectious plasma of a
chimp. Kuo et al., Science 244: 359-361 (1989); Choo et al., Science 244:
362-364 (1989). cDNA (copy DNA) sequences from HCV were identified which
encode antigens that react immunologically with antibodies present in a
majority of the patients clinically diagnosed with NANBH. Based on the
information available and on the molecular structure of HCV, the genetic
makeup of the virus consists of single stranded linear RNA (positive
strand) of molecular weight approximately 9.5 kb, and possessing one
continuous translational open reading frame. J. A. Cuthbert, Amer. J. Med.
Sci. 299: 346-355 (1990). It is a small enveloped virus resembling the
Flaviviruses. Investigators have made attempts to identify the NANB agent
by ultrastructural changes in hepatocytes in infected individuals. H.
Gupta, Liver 8: 111-115 (1988 ); D. W. Bradley J. Virol. Methods 10:
307-319 (1985). Similar ultrastructural changes in hepatocytes as well as
PCR amplified HCV RNA sequences have been detected in NANBH patients as
well as in chimps experimentally infected with infectious HCV plasma. T.
Shimizu et al., Proc. Natl. Acad. Sci. 87: 6441-6444 (1990).
Considerable serological evidence has been found to implicate HCV as the
etiological agent for post-transfusion NANBH. H. Alter et al., N. Eng. J.
Med. 321: 1494-1500 (1989); Estaben et al., The Lancet: August 5: 294-296
(1989); C. Van Der Poel et al., The Lancet August 5: 297-298 (1989); G.
Sbolli, J. Med. Virol. 30: 230-232 (1990); M. Makris et al., The Lancet
335: 1117-1119 (1990). Although the detection of HCV antibodies eliminates
70 to 80% of NANBH infected blood from the blood supply system, the
antibodies apparently are readily detected during the chronic state of the
disease, while only 60% of the samples from the acute NANBH stage are HCV
antibody positive. H. Alter et al., New Eng. J. Med. 321: 1994-1500
(1989). These data clearly indicated the need for the identification of
additional HCV proteins for efficient serodiagnosis of HCV infection.
Following the cloning and expression of structural protein CORE and 33C,
second generation antibody assays have been developed which employ HCV
CORE and 33C proteins in addition to C-100 for the detection of antibodies
to HCV in NANB patients. Although the second generation assays have
significantly increased the sensitivity of detection, the prolonged
interval between exposure to HCV and antibody detection, and the lack of
adequate information regarding the profile of immune response to various
structural and non-structural proteins raises questions regarding the
infectious state of the patient in the antibody negative phase during
NANBH infection. Therefore, there is a need for the development of assay
systems to identify acute infection to HCV and the presence of HCV.
SUMMARY OF THE INVENTION
The present invention provides a panel of highly specific and novel
monoclonal antibodies that can be employed for the detection of putative
HCV E2/NS1 antigens. The monoclonal antibodies specifically bind to
protein sequences derived from the putative HCV E2/NS1 gene. The
hybridomas which produce these monoclonal antibodies are identified as
follows: hybridoma H13C113 (A.T.C.C. deposit No. HB 10856) and hybridoma
H23C163 (A.T.C.C. deposit No. HB 10857).
The specificity of these monoclonal antibodies enables the advantageous
identification of HCV antigen in the putative E2/NS1 region, which
identification can be useful in differentiation studies as well as in the
diagnosis and evaluation of HCV (NANB) infections.
In a preferred assay format, a test sample which may contain HCV antigens
is contacted with a solid phase to which a polyclonal or a monoclonal
anti-HCV E2/NS1 antibody or a fragment thereof has been bound, to form a
mixture. This mixture is incubated for a time and under conditions
sufficient for antigen/antibody complexes to form. The so-formed complexes
then are contacted with an indicator reagent comprising a monoclonal or
polyclonal antibody or a fragment thereof, specific for the HCV antigen
attached to a signal generating compound to form a second mixture. This
second mixture is reacted for a time and under conditions sufficient to
form antibody/antigen/antibody complexes. The presence of HCV antigen is
determined by detecting the measurable signal generated. The amount of HCV
present in the test sample, thus the amount of HCV antigen captured on the
solid phase, is proportional to the amount of signal generated.
Alternatively, an indicator reagent comprising a monoclonal or polyclonal
antibody, or fragment thereof, specific for HCV E2/NS1 antigen and a
signal generating compound is added to a polyclonal or monoclonal anti-HCV
antibody or fragment thereof coated on a solid phase and the test sample,
to form a mixture. This mixture is incubated for a time and under
conditions sufficient to form antibody/antigen/antibody complexes. The
presence and amount of HCV present in the test sample, and thus the amount
of HCV antigen captured on the solid phase, is determined by detecting the
measurable signal. The amount of HCV present in the test sample is
proportional to the amount of signal generated.
In another alternate assay format, one or a combination of more than one
monoclonal antibody of the invention can be employed as a competitive
probe for the detection of antibodies to HCV E2/NS1 antigen. For example,
HCV E2/NS1 antigens, either alone or in combination, can be coated on a
solid phase. A test sample suspected of containing antibody to HCV E2/NS1
antigen then is incubated with an indicator reagent comprising a signal
generating compound and a monoclonal antibody of the invention for a time
and under conditions sufficient to form antigen/antibody complexes of
either the test sample and indicator reagent to the solid phase or the
indicator reagent to the solid phase. The reduction in binding of the
monoclonal antibody to the solid phase can be quantitatively measured. A
measurable reduction in the signal compared to the signal generated from a
confirmed negative NANBH test sample would indicate the presence of
anti-HCV E2/NS1 antibody in the test sample.
In yet another assay format, a test sample is contacted with a solid phase
to which HCV E2/NS1 proteins are attached and an indicator reagent
comprising a monoclonal antibody or fragment thereof specific for HCV
E2/NS1 attached to a signal generating compound, to form a mixture. The
mixture is incubated for a time and under conditions sufficient for
antibody/antigen complexes to form. The presence of anti-HCV present in
the test sample is determined by detecting the measurable signal
generated, and comparing the signal to the measured signal generated from
a known negative sample. A measurable reduction of signal of the test
sample, compared to the known negative sample's signal, is indicative of
the presence of anti-HCV antibodies. Competitive assays for the detection
of anti-HCV antibody using antigens free in solution also can be
performed.
The presence of HCV E2/NS1 antigen can be detected in a tissue sample by
contacting the tissue sample with an indicator reagent comprising a signal
generating compound attached to a monoclonal antibody which specifically
binds to HCV E2/NS1 antigen or fragment thereof, to form a mixture. This
mixture is incubated for a time and under conditions sufficient for
antigen/antibody complex to form. The presence of HCV E2/NS1 antigen
present in the tissue sample is determined by detecting the signal
generated.
Also provided are kits useful for determining the presence of HCV NS1
antigen or antibody in test samples that include the monoclonal antibodies
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration of the location of the recombinant HCV proteins
on the HCV genome employed either as immunogens for the generation of
monoclonal antibodies or for their characterization.
FIG. 2 is a Western blot analysis illustrating specific binding monoclonal
antibodies H13C113 and H23C163 to HCV NS1.
FIG. 3 is a profile of PEPSCAN analysis with overlapping hexamer peptides
(a.a. 600-720 of HCV) of monoclonal antibody H13C113 illustrating the
epitope specificity of H13C113 to HCV a.a. 649-655.
FIG. 4 is a profile of PEPSCAN analysis with overlapping hexamer peptides
(a.a. 600-720 of HCV) of monoclonal antibody H23C163 illustrating the
epitope specificity of H23C163 to HCV a.a. 649-655.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel monoclonal antibodies to the putative
HCV E2/NS1 protein, methods for using the monoclonal antibodies, and kits
which contain these monoclonal antibodies.
The monoclonal antibodies of the present invention can be employed in
various assay systems to determine the presence, if any, of HCV E2/NS1
proteins in a test sample. Fragments of these monoclonal antibodies
provided also may be used. For example, in a first assay format, a
polyclonal or monoclonal anti-HCV E2/NS1 antibody or fragment thereof, or
a combination of these antibodies, which has been coated on a solid phase,
is contacted with a test sample which may contain HCV E2/NS1 proteins, to
form a mixture. This mixture is incubated for a time and under conditions
sufficient to form antigen/antibody complexes. Then, an indicator reagent
comprising a monoclonal or a polyclonal antibody or a fragment thereof,
which specifically binds to the HCV E2/NS1 region, or a combination of
these antibodies, to which a signal generating compound has been attached,
is contacted with the antigen/antibody complexes to form a second mixture.
This second mixture then in incubated for a time and under conditions
sufficient to form antibody/antigen/antibody complexes. The presence of
HCV E2/NS1 antigen present in the test sample and captured on the solid
phase, if any, is determined by detecting the measurable signal generated
by the signal generating compound. The amount of HCV E2/NS1 antigen
present in the test sample is proportional to the signal generated.
Alternatively, a polyclonal or monoclonal anti-HCV E2/NS1 antibody or
fragment thereof, or a combination of these antibodies which is bound to a
solid support, the test sample and an indicator reagent comprising a
monoclonal or polyclonal antibody or fragments thereof, which specifically
binds to HCV E2/NS1 antigen, or a combination of these antibodies to which
a signal generating compound is attached, are contacted to form a mixture.
This mixture is incubated for a time and under conditions sufficient to
form antibody/antigen/antibody complexes. The presence, if any, of HCV
E2/NS1 proteins present in the test sample and captured on the solid phase
is determined by detecting the measurable signal generated by the signal
generating compound. The amount of HCV proteins present in the test sample
is proportional to the signal generated.
In another alternate assay format, one or a combination of one or more
monoclonal antibodies of the invention can be employed as a competitive
probe for the detection of antibodies to HCV protein. For example, HCV
proteins, either alone or in combination, can be coated on a solid phase.
A test sample suspected of containing antibody to HCV E2/NS1 antigen then
is incubated with an indicator reagent comprising a signal generating
compound and at least one monoclonal antibody of the invention for a time
and under conditions sufficient to form antigen/antibody complexes of
either the test sample and indicator reagent to the solid phase or the
indicator reagent to the solid phase. The reduction in binding of the
monoclonal antibody to the solid phase can be quantitatively measured. A
measurable reduction in the signal compared to the signal generated from a
confirmed negative NANBH test sample indicates the presence of anti-HCV
E2/NS1 antibody in the test sample.
In yet another detection method, each of the monoclonal antibodies of the
present invention can be employed in the detection of HCV antigens in
fixed tissue sections, as well as fixed cells by immunohistochemical
analysis.
In addition, these monoclonal antibodies can be bound to matrices similar
to CNBr-activated Sepharose and used for the affinity purification of
specific HCV proteins from cell cultures, or biological tissues such as
blood and liver.
The monoclonal antibodies of the invention can also be used for the
generation of chimeric antibodies for therapeutic use, or other similar
applications.
The monoclonal antibodies or fragments thereof can be provided individually
to detect HCV E2/NS1 antigens. It is contemplated that combinations of the
monoclonal antibodies (and fragments thereof) provided herein also may be
used together as components in a mixture or "cocktail" of at least one
anti-HCV E2/NS1 antibody of the invention with antibodies to other HCV
regions, each having different binding specificities. Thus, this cocktail
can include the monoclonal antibodies of the invention which are directed
to HCV E2/NS1 proteins and other monoclonal antibodies to other antigenic
determinants of the HCV genome. Examples of other monoclonal antibodies
useful for these contemplated cocktails include those to HCV C-100, HCV
33C, HCV CORE, HCV NS5 and/or HCV putative ENV, which are disclosed in,
for example, U.S. Ser. No. 07/610,175 entitled MONOCLONAL ANTIBODIES TO
HEPATITIS C VIRUS AND METHOD FOR USING SAME, U.S.S.N. 07/610,175 entitled
MONOCLONAL ANTIBODIES TO HCV 33C PROTEINS AND METHODS FOR USING SAME, U.S.
Ser. No. 07/648,475 entitled MONOCLONAL ANTIBODIES TO PUTATIVE HCV
ENVELOPE REGION AND METHODS FOR USING SAME, U.S. Ser. No. 07/648,473
entitled MONOCLONAL ANTIBODIES TO HCV CORE PROTEINS AND METHODS FOR USING
SAME and in co-filed patent application entitled MONOCLONAL ANTIBODIES TO
HCV NS5 PROTEIN AND METHODS FOR USING SAME, U.S. Ser. No. 07/748,563, all
of which enjoy common ownership and are incorporated herein by reference.
This cocktail of monoclonal antibodies as described herein would be used
in the assay formats detailed herein in place of the monoclonal antibody
to HCV E2/NS1, and thus would be able to detect the E2/NS1 and other HCV
antigens.
The polyclonal antibody or fragment thereof which can be used in the assay
formats should specifically bind to HCV putative E2/NS1 region or other
HCV proteins used in the assay, such as HCV C-100 protein, HCV 33C
protein, HCV CORE, HCV ENV or HCV NS5 protein. The polyclonal antibody
used preferably is of mammalian origin; human, goat, rabbit or sheep
anti-HCV polyclonal antibody can be used. Most preferably, the polyclonal
antibody is rabbit polyclonal anti-HCV antibody. The polyclonal antibodies
used in the assays can be used either alone or as a cocktail of polyclonal
antibodies. Since the cocktails used in the assay formats are comprised of
either monoclonal antibodies or polyclonal antibodies having different HCV
specificity, they would be useful for diagnosis, evaluation and prognosis
of HCV infection, as well as for studying HCV protein differentiation and
specificity.
Test samples which can be tested by the methods of the present invention
described herein include human and animal body fluids such as whole blood,
serum, plasma, cerebrospinal fluid, urine, biological fluids such as cell
culture supernatants, fixed tissue specimens and fixed ceil specimens.
The "solid phase" is not critical and can be selected by one skilled in the
art. Thus, latex particles, microparticles, magnetic or non-magnetic
beads, membranes, plastic tubes, walls of microtiter wells, glass or
silicon chips and sheep red blood cells are all suitable examples.
Suitable methods for immobilizing peptides on solid phases include ionic,
hydrophobic, covalent interactions and the like. A "solid phase", as used
herein, refers to any material which is insoluble, or can be made
insoluble by a subsequent reaction. The solid phase can be chosen for its
intrinsic ability to attract and immobilize the capture reagent.
Alternatively, the solid phase can retain an additional receptor which has
the ability to attract and immobilize the capture reagent. The additional
receptor can include a charged substance that is oppositely charged with
respect to the capture reagent itself or to a charged substance conjugated
to the capture reagent. As yet another alternative, the receptor molecule
can be any specific binding member which is immobilized upon (attached to)
the solid phase and which has the ability to immobilize the capture
reagent through a specific binding reaction. The receptor molecule enables
the indirect binding of the capture reagent to a solid phase material
before the performance of the assay or during the performance of the
assay. The solid phase thus can be a plastic, derivatized plastic,
magnetic or non-magnetic metal, glass or silicon surface of a test tube,
microtiter well, sheet, bead, microparticle, chip, and other
configurations known to those of ordinary skill in the art.
It is contemplated and within the scope of the invention that the solid
phase also can comprise any suitable porous material with sufficient
porosity to allow access by detection antibodies and a suitable surface
affinity to bind antigens. Microporous structures are generally preferred,
but materials with gel structure in the hydrated state may be used as
well. Such useful solid supports include:
natural polymeric carbohydrates and their synthetically modified,
cross-linked or substituted derivatives, such as agar, agarose,
cross-linked alginic acid, substituted and cross-linked guar gums,
cellulose esters, especially with nitric acid and carboxylic acids, mixed
cellulose esters, and cellulose ethers;
natural polymers containing nitrogen, such as proteins and derivatives,
including cross-linked or modified gelatins;
natural hydrocarbon polymers, such as latex and rubber;
synthetic polymers which may be prepared with suitably porous structures,
such as vinyl polymers, including polyethylene, polypropylene,
polystyrene, polyvinylchloride, polyvinylacetate and its partially
hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and
terpolymers of the above polycondensates, such as polyesters, polyamides,
and other polymers, such as polyurethanes or polyepoxides;
porous inorganic materials such as sulfates or carbonates of alkaline earth
metals and magnesium, including barium sulfate, calcium sulfate, calcium
carbonate, silicates of alkali and alkaline earth metals, aluminum and
magnesium; and aluminum or silicon oxides or hydrates, such as clays,
alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may
be used as filters with the above polymeric materials); and
mixtures or copolymers of the above classes, such as graft copolymers
obtained by initializing polymerization of synthetic polymers on a
pre-existing natural polymer. All of these materials may be used in
suitable shapes, such as films, sheets, or plates, or they may be coated
onto or bonded or laminated to appropriate inert carriers, such as paper,
glass, plastic films, or fabrics.
The porous structure of nitrocellulose has excellent absorption and
absorption qualities for a wide variety of reagents including monoclonal
antibodies. Nylon also possesses similar characteristics and also is
suitable.
It is contemplated that such porous solid supports described hereinabove
are preferably in the form of sheets of thickness from about 0.01 to 0.5
mm, preferably about 0.1 mm. The pore size may vary within wide limits,
and is preferably from about 0.025 to 15 microns, especially from about
0.15 to 15 microns. The surfaces of such supports may be activated by
chemical processes which cause covalent linkage of the antigen or antibody
to the support. The irreversible binding of the antigen or antibody is
obtained, however, in general, by adsorption on the porous material by
poorly understood hydrophobic forces. Suitable solid supports also are
described in U.S. patent application Ser. No. 227,272.
The indicator reagent comprises a signal generating compound (label) which
is capable of generating a measurable signal detectable by external means
conjugated (attached) to a specific binding member for HCV. "Specific
binding member" as used herein means a member of a specific binding pair.
That is, two different molecules where one of the molecules through
chemical or physical means specifically binds to the second molecule. In
addition to being an antibody member of a specific binding pair for HCV,
the indicator reagent also can be a member of any specific binding pair,
including either hapten-anti-hapten systems such as biotin or anti-biotin,
avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide
sequence, an effector or a receptor molecule, an enzyme cofactor and an
enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive
specific binding member can be an antibody, an antigen, or an
antibody/antigen complex that is capable of binding either to HCV as in a
sandwich assay, to the capture reagent as in a competitive assay, or to
the ancillary specific binding member as in an indirect assay.
The various signal generating compounds (labels) contemplated include
chromogens, catalysts such as enzymes, luminescent compounds such as
fluorescein and rhodamine, chemiluminescent compounds such as acridinium,
phenanthridinium and dioxetane compounds, radioactive elements, and direct
visual labels. Examples of enzymes include alkaline phosphatase,
horseradish peroxidase, beta-galactosidase, and the like. The selection of
a particular label is not critical, but it will be capable of producing a
signal either by itself or in conjunction with one or more additional
substances.
Other embodiments which utilize various other solid phases also are
contemplated and are within the scope of this invention. For example, ion
capture procedures for immobilizing an immobilizable reaction complex with
a negatively charged polymer, described in co-pending U.S. patent
application Ser. No. 150,278 corresponding to EP publication 0326100, and
U.S. patent application Ser. No. 375,029 (EP publication no. 0406473) both
of which enjoy common ownership and are incorporated herein by reference,
can be employed according to the present invention to effect a fast
solution-phase immunochemical reaction. An immobilizable immune complex is
separated from the rest of the reaction mixture by ionic interactions
between the negatively charged poly-anion/immune complex and the
previously treated, positively charged porous matrix and detected by using
various signal generating systems previously described, including those
described in chemiluminescent signal measurements as described in
co-pending U.S. patent application Ser. No. 921,979 corresponding to EPO
Publication No. 0 273,115, which enjoys common ownership and which is
incorporated herein by reference.
Also, the methods of the present invention can be adapted for use in
systems which utilize microparticle technology including in automated and
semi-automated systems wherein the solid phase comprises a microparticle.
Such systems include those described in pending U.S. patent application
Ser. Nos. 425,651 and 425,643, which correspond to published EPO
applications Nos. EP 0 425 633 and EP 0 424 634, respectively, which are
incorporated herein by reference.
The use of scanning probe microscopy (SPM) for immunoassays also is a
technology to which the monoclonal antibodies of the present invention are
easily adaptable. In scanning probe microscopy, in particular in atomic
force microscopy, the capture phase, for example, at least one of the
monoclonal antibodies of the invention, is adhered to a solid phase and a
scanning probe microscope is utilized to detect antigen/antibody complexes
which may be present on the surface of the solid phase. The use of
scanning tunnelling microscopy eliminates the need for labels which
normally must be utilized in many immunoassay systems to detect
antigen/antibody complexes. Such a system is described in pending U.S.
patent application Ser. No. 662,147, which enjoys common ownership and is
incorporated herein by reference.
The use of SPM to monitor specific binding reactions can occur in many
ways. In one embodiment, one member of a specific binding partner (the
analyte specific substance, which is the monoclonal antibody of the
invention) is attached to a surface suitable for scanning. The attachment
of the analyte specific substance may be by adsorption to a test piece
which comprises a solid phase of a plastic or metal surface, following
methods known to those of ordinary skill in the art. Or, covalent
attachment of a specific binding partner (analyte specific substance) to a
test piece which test piece comprises a solid phase of derivatized
plastic, metal, silicon, or glass may be utilized. Covalent attachment
methods are known to those skilled in the art and include a variety of
means to irreversibly link specific binding partners to the test piece. If
the test piece is silicon or glass, the surface must be activated prior to
attaching the specific binding partner. Activated silane compounds such as
triethoxy amino propyl silane (available from Sigma Chemical Co., St.
Louis, Mo.), triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee,
Wis.), and (3-mercapto-propyl)trimethoxy silane (Sigma Chemical Co., St.
Louis, Mo.) can be used to introduce reactive groups such as amino-,
vinyl, and thiol, respectively. Such activated surfaces can be used to
link the binding partner directly (in the cases of amino or thiol) or the
activated surface can be further reacted with linkers such as
glutaraldehyde, bis(succinimidyl) suberate, SPPD 9 succinimidyl
3-[2-pyridyldithio] propionate), SMCC (succinimidyl-4-[N-maleimidomethyl]
cyclohexane-1-carboxylate), SIAB (succinimidyl [4-iodoacetyl]
aminobenzoate), and SMPB (succinimidyl 4-[1-maleimidophenyl] butyrate) to
separate the binding partner from the surface. The vinyl group can be
oxidized to provide a means for covalent attachment. It also can be used
as an anchor for the polymerization of various polymers such as poly
acrylic acid, which can provide multiple attachment points for specific
binding partners. The amino surface can be reacted with oxidized dextrans
of various molecular weights to provide hydrophilic linkers of different
size and capacity. Examples of oxidizable dextrans include Dextran T-40
(molecular weight 40,000 daltons), Dextran T-110 (molecular weight 110,000
daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M
(molecular weight 2,000,000 daltons) (all of which are available from
Pharmacia, Piscataway, N.J.), or Ficoll (molecular weight 70,000 daltons
(available from Sigma Chemical Co., St. Louis, Mo.). Also, polyelectrolyte
interactions may be used to immobilize a specific binding partner on a
surface of a test piece by using techniques and chemistries described by
pending U.S. patent application Ser. Nos. 150,278, filed Jan. 29, 1988,
and Ser. No. 375,029, filed Jul. 7, 1989, each of which enjoys common
ownership and each of which is incorporated herein by reference. The
preferred method of attachment is by covalent means. Following attachment
of a specific binding member, the surface may be further treated with
materials such as serum, proteins, or other blocking agents to minimize
non-specific binding. The surface also may be scanned either at the site
of manufacture or point of use to verify its suitability for assay
purposes. The scanning process is not anticipated to alter the specific
binding properties of the test piece.
While the present invention discloses the preference for the use of solid
phases, it is contemplated that the monoclonal antibodies of the present
invention can be utilized in non-solid phase assay systems. These assay
systems are known to those skilled in the art, and are considered to be
within the scope of the present invention.
It is contemplated that the reagent employed for the assay can be provided
in the form of a kit with one or more containers such as vials or bottles,
with each container containing a separate reagent such as a monoclonal
antibody, or a cocktail of monoclonal antibodies, detection reagents and
washing reagents employed in the assay.
The following examples demonstrate the advantages and utility of this
invention for serodiagnosis of HCV by describing methods for the
development, characterization, epitope mapping and clinical utility of
these monoclonal antibodies. The methods used for monoclonal antibody
development follow procedures known in the art and detailed in Kohler and
Milstein, Nature 256: 494 (1975) and reviewed in J. G. R. Hurrel, ed.,
Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,
Inc., Boca Raton, Fla. (1982). Another method of monoclonal antibody
development which is based on the Kohler and Milstein method is that of L.
T. Mimms et al., Virology 176: 604-619 (1990), which is incorporated
herein by reference. These examples are meant to illustrate, but not to
limit, the spirit and scope of the invention.
EXAMPLES
EXAMPLE 1
Immunization of Mice with SEQ. ID. No. 6 Selection of Synthetic Peptide for
Generation of Monoclonal Antibodies to HCV E2/NS1 Region
Immunogenic domains of E2/NS1 region of HCV genome encompassing a.a.
600-720 (SEQ. ID. NO. 1) were mapped with PEPSCAN analysis. A PEPSCAN kit
was purchased from Cambridge Research Bioscience (Valley Stream, N.Y.,
U.S.A.) to synthesize a series of overlapping hexamer peptides (overlap of
five amino acids) encompassing HCV a.a. 600-720 (SEQ. ID. NO. 1), on
derivitized polypropylene pins supplied by the manufacturer. The synthesis
protocol supplied with the kit was followed exactly for the synthesis of
these peptides. Briefly, the polypropylene pins which contained the F-moc
.beta.-alanine as the end group amino acid were deprotected with 20% (v/v)
piperidine in dimethylformamide (DMF) for 30 min. Pins were washed with
DMF (1.times.5 min.), Methanol (4.times.2 min.) followed by a final DMF
wash (1.times.5 min.). F-moc active esters of amino acids were prepared at
30 mM concentration in 1-hydroxybenzotriazole (HOBt) in DMF. Amino acids
were dispensed (175 ul) in wells of 96 well microtiter plates supplied
with the kit in desired sequence, starting at the carboxy terminus.
Deprotected pins were lowered in the amino acid solutions and incubated at
room temperature (RT) overnight. Following the DMF methanol wash sequence
as described above, the deprotection, washing and coupling steps were
repeated until all amino acid in each of the peptides sequence were
coupled. After a final deprotection step, the terminal amino acids were
acetylated by incubating the pins with DMF:acetic anhydride:triethylamine
at 5:2:1 (v/v/v) for 90 min. at RT. Following the DMF/methanol wash
sequence, pins were air dried. Before the serological analysis, the final
side chain deprotection and neutralization was accomplished by treating
the pins with Trifluoroacetic acid:Phenol:Ethanedithiol at 95:2.5:2.5
(v/w/v). Pins were washed with dichloromethane (2.times.2 min.), 5%
diisopropylethylamine/dichloromethane (1.times.5 min.) and dichloromethane
(1.times.5 min.). Finally, pins were air dried, washed with water, soaked
in methanol for 18 hrs., dried and stored dessicated in refrigerator.
FAB dimers of IgG purified from sera of individuals seropositive for
antibodies to HCV proteins were used as the primary antibody for the
serological analysis of these peptides using the EIA procedure recommended
by the manufacturer. Briefly, the primary antibody was diluted to
appropriate concentration in phosphate buffered saline (PBS) containing
0.1% Tween-20.RTM. (Bio-Rad, Richmond, Calif.), 1% ovalbumin (available
from Sigma, St. Louis, Mo.), and 1% bovine serum albumin (available from
Sigma). Peptide pins were incubated with the primary antibody overnight at
4.degree. C. Following several washes with PBS/Tween-20.RTM., pins were
incubated with appropriately diluted goat anti-mouse HRPO for 1 hr. at
room temperature. Azido-di-3-ethylbenzthiazodinsulphonate dissolved in a
phosphate-citrate buffer containing hydrogen peroxide was used as the
color developing reagent. The optical density of the color developed was
measured at 405 nm after incubation of the pins with the developing
reagent for 15-20 min. Based on the reactivity of these sera in EIA, four
amino acid sequences (a.a. 607-627 (SEQ. ID. No. 2), a.a. 643-663 (SEQ.
ID. No. 3), a.a. 666-683 (SEQ. ID. No. 4) and a.a. 671-691 (SEQ. ID. NO. 5
were identified as the immunogenic domains as disclosed in U.S. patent
application Ser. No. 610,180 previously incorporated herein by reference.
Each of these four sequences and an additional sequence, which was the
combination of the two most immunogenic sequences (a.a. 643-683) (SEQ. ID.
No. 6) were synthesized by a stepwise solid phase synthesis starting at
the carboxy terminus by a procedure similar to that described in E. Gross
and T. Heinhofer, eds. Barany and Merrifield, The Peptides 2: 1284,
Academic Press, New York, N.Y. Based on the EIA reactivity of a panel of
HCV positive sera as disclosed in the U.S. patent application Ser. No.
610,180 previously incorporated herein by reference, peptide 643-683 (SEQ.
ID. NO. 6) was chosen as the immunogen for the generation of monoclonal
antibodies to HCV NS1. FIG. 1 shows the location of these peptides on the
HCV genome.
Immunization of Mice
Female Balb/c were immunized with approximately 50 ug of the crude peptide
643-683 (HCV a.a. 643-683, SEQ. ID. NO. 6) using the RIBI adjuvant system
(RIBI Immunochemicals Res., U.S.A.). On day one, mice received 50 ug of
the peptide with 50 ug each of Trehalose dimycolate (TDM) and M. Phlei in
a buffer emulsion prepared according to the manufacturer's instructions.
Subsequent immunizations were done on day 18, 34, 42 and 63. Mice were
bled on day 25 and 77, and the immune response was assessed by EIA using
microtiter plates coated with the immunogen. Mice were allowed to rest for
at least eight weeks before the fusion.
Enzyme-Linked Immunoassay (EIA)
The immune response to the immunizing antigen was assessed by microtiter
EIA. Wells of microtiter plates were coated with 100 .mu.l of purified
synthetic peptide (a.a. 643-683, SEQ. ID. NO. 6) on 0.1M bicarbonate
buffer at pH 9.5. After washing with Phosphate Buffered Saline (PBS) which
also contained 0.01% sodium dodecyl sulfate (SDS) and 0.05% Tween-20.RTM.
(available from Bio-Rad Laboratories, Richmond, Calif.) free sites were
overcoated with 1% BSA in bicarbonate buffer at pH 9.5. Plates were stored
at 4.degree. C. following a final wash. Sera from native or immunized mice
were serially diluted in 100 .mu.l of dilution buffer which contained 20
mM sodium phosphate, pH 7.4, 0.15M NaCl, 20% normal goat serum, 10% fetal
calf serum, 5 mM EDTA, 10 mM EGTA, 50 mM Tris, 0.2% Tween-20.RTM. with
sodium azide as a preservative (at pH 6.8). The diluted sera were reacted
with the antigen for three (3) hours at 37.degree. C. The plates were
washed and 100 .mu.l of appropriately diluted goat anti-mouse IgG (heavy
[h] and light [l] chain) Horseradish Peroxidase (HRPO)-conjugated antibody
(Jackson Immunochemicals, West Grove, Pa.) was added. The plates were
incubated at 37.degree. C. for two (2) hours. After a final wash, 100
.mu.l of o-phenylenediamine:2 HCL (OPD) color reagent was added. The
reaction was carried out at room temperature for 10 to 30 minutes, and
then stopped by the addition of 1 ml of 1N H.sub.2 SO.sub.4. The
absorbance at 492/600 nm was recorded, which was found to be directly
proportional to the amount of specific antibody bound to the antigen.
EXAMPLE 2
Cell Fusion
Upon demonstration of specific anti-HCV antibody present at reasonable
titers in sera of immunized mice, the mice were allowed to rest for at
least eight weeks prior to a pre-fusion boost of antigen. The pre-fusion
antigen boost then was performed by intravenous (IV) tail vein injection
of approximately 40 .mu.g of respective purified HCV synthetic peptide
(SEQ. ID. NO. 6). Three days later the mice were sacrificed, and their
spleens which contained anti-HCV antibody-producing cells were disrupted
into single cells. These single cell suspensions were treated with 0.83%
NH.sub.4 Cl to remove red blood cells, and then these suspensions were
mixed with SP2/0 cells at a 10:1 (SP2/0:spleen cells) ratio. The mixed
cells were centrifuged, washed once with serum-free medium, and again
centrifuged. The fusogen polyethylene glycol (PEG) was used to form
hybrids of the immune donor spleen cells with the myeloma cell line SP2/0
(HRPT neg.). Kohler and Milstein, Nature 356:494 (1975), and reviewed in
J. G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques and
Applications, CRC Press, Inc., Boca Raton, Fla. (1982). Briefly, fusion of
the spleen and SP2/0 cells was accomplished by exposing the pellet to 40%
PEG (ATCC, mw 1300-1600) In serum-free Iscoe's Modified Dulbecco's Medium
(IMDM) for two minutes. The PEG and cell suspension was diluted slowly by
the addition of 20 ml of serum-free IMDM over a period of five minutes,
followed by collection of the cells by centrifugation. The supernatant was
decanted and replaced with 30 ml IMDM containing 20% fetal bovine serum
(FBS) (Hyclone Laboratories, Logan, Utah) with HAT (hypoxanthine,
aminopterin and thymidine) media in order to select for hybridomas. Spleen
cells from one non-immune BABB/c mouse also were added as a feeder layer.
The cells were plated at 0.1 ml/well in three 96-well tissue culture
plates. An additional 0.1 ml of HAT media was added to each well three
days later. At weekly intervals thereafter, one-half the media was
replaced with IMDM containing 20% FBS with HT (hypoxanthine and
thymidine), and hybrids were allowed to grow for an additional seven to
fourteen days.
It was found that some of the hybrids were composed of spleen cells making
antibody to HCV fused with SP2/0 cells. Briefly, the fusogen promoted
fusion of spleen cell and SP2/0 cell membranes, which formed a
heterokaryon containing nuclei of both cells. Eventually, the dissimilar
nuclei fuse produced a single nucleus capable of synchronous mitosis. As
the fused cells divided, the hybrid stabilized by losing chromosomes of
each nucleus. The fused cells were plated into multiple 96-well plates at
10.sup.5 to 10.sup.6 cells per wall. The hybrid cells formed from
SP2/0:spleen cell fusions were selectively propagated by culturing in HAT
medium. All unused SP2/0 or SP2/0:SP2/0 fused cells were prevented from
growing aminopterin, and unfused spleen cells on spleen:spleen fused cells
died off in culture. Only SP2/0:spleen cell hybrids grew in the HAT
selective medium.
EXAMPLE 3
Screening and Cloning of Monoclonal Antibodies
After 10 to 14 days, culture fluids from wells containing hybridoma cell
growth were screened for the presence of a monospecific antibody as
follows. Each of the hybridoma supernatants from the NS1 fusions were
tested by the EIA procedure described in Example 1 with the synthetic
peptide a.a. 643-683 (SEQ. ID. NO. 6) coated on the solid phase. Hybridoma
culture fluids reacting specifically to the immunogen, i.e., HCV protein
SEQ. ID. NO. 6 were selected for cloning by the limiting dilution method,
using the guidelines outlines by J. W. Goding, Monoclonal Antibodies:
Principles and Practices, Academic Press, New York (1983). Culture
supernatant of cloned samples were tested again by EIA with the immunogen
as described above in Example 1, for the confirmation of monospecific
reactivity to HCV protein sequence. Clones with strongest reactivity
specifically to the synthetic peptide were selected for expansion and
further analysis.
EXAMPLE 4
Amplification of Antibody Yields by Ascites Method
In order to obtain greater amounts of monoclonal antibodies, 10 to 20
million cloned cells of the desired hybridoma cell line were inoculated
into a BALB/c mouse previously treated i.p. with 0.5 ml pristane
(2,6,10,14-tetramethylpentadecane) by the method outlined in J. G. R.
Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques and Applications,
CRC Press, Inc., Boca Raton, Fla. (1982). Pristane treatment enhanced
growth of mouse myeloma hybrids within the peritoneum of the mouse, and
the ascites fluids which formed were rich in the monoclonal antibody
secreted by the hybrid cells. After formation of adequate ascites fluid
(approximately seven days), the mice were sacrificed and the ascites were
withdrawn from the peritoneum, clarified by centrifugation and stored at
-20.degree. C. Monoclonal antibodies from ascites fluid were purified
using protein-A sepharose (according to J. G. R. Hurrell ed., supra). All
characterization procedures described herein were performed with either
culture supernatants, ascites fluids or protein-A purified IgG.
EXAMPLE 5
Characterization of Monoclonal Antibodies EIA
Purified IgG of monoclonal antibodies were titrated on microtiter plates
coated with the immunogen (peptide 643-683, SEQ, ID. NO. 6) as well as on
plates coated with the purified recombinant HCV E2/NS1 protein PHCV80
(a.a. 365-731, SEQ ID NO. 7) by the EIA protocol described in Example 1.
The detail description of cloning and expression of pHCV80 is described in
Example 6. EIA reactivity of monoclonal antibodies of this invention to
the immunogen as well as the recombinant HCV E2/NS1 protein is described
in Table 1.
Western Blot Analysis
Approximately 300 .mu.g of the HCV protein PHCV-80 (a.a. 365-731, SEQ. ID.
NO. 7) were treated with SDS and 2-mercaptoethanol at 95.degree. C., and
electrophoresed in a 12% polyacrylamide-SDS gel (Laemmli et al., Nature
227:680-685 (1970). Proteins were transferred overnight from the gel to
nitrocellulose by electrophoresis at 100 mamp, or transferred in 1-2 hours
at 1.0 amp, in a standard transfer buffer which comprised 25 mM Tris
[(Hydroxymethyl) Aminomethane] 192 mM glycine, and 2.0% methanol, pH 8.3.
(Towbin et al., Proc. Natl. Acad. Sci. 73:4350-4354 [1979]). After
transferring the proteins and blocking the nitrocellulose with 5% dry milk
in PBS, the nitrocellulose was cut into strips (each strip containing
approximately 5 .mu.g of the protein which then were used to determine the
presence of anti-HCV antibody in test sera (or other samples). Reaction
mixtures consisted of a nitrocellulose strip incubated with an appropriate
amount of test sample in 2.0 ml of buffer (20 mM Tris, 1 mM EDTA, 0.2M
NaCl, 0.3% Triton X-100.RTM. and 2 mg/ml bovine serum albumin (BSA), pH
7.5, 5% E. Coli lysate and 3% CKS lysate overnight at 4.degree. C. The
strips were washed with buffered detergent (10 mM phosphate buffered
saline (PBS) pH 7.5, containing 0.1% SDS and 0.5% Triton X-100.RTM.)
followed by addition of goat anti-mouse IgG antibody conjugated to HRPO.
The strips were incubated for one to two hours at room temperature,
followed by washing with buffered detergent. Finally, antibody bound to
the protein was visualized by addition of freshly prepared HRP color
reagent (Bio-Rad Laboratories, Richmond CA) (120 mg dissolved in 40 ml
ice-cold methanol, then diluted into 200 ml Tris buffered saline [TBS] pH
7.8, containing 120 .mu.l of 30% hydrogen peroxide. FIG. 2 illustrates the
specific reactivity of the monoclonals of this invention to the HCV E2/NS1
protein.
Competition with Immune Human Sera
In order to establish whether each of the monoclonal antibodies recognized
an epitope that is immunologic in humans, a competition assay was
performed as follows. Each of the monoclonal antibodies was tested in an
assay where the monoclonal antibody competed with a human sera
seropositive for antibody to E2/NS1 (SEQ. ID. NO. 1) for the binding to
the antigen. Briefly, a human serum from an individual infected with NANBH
and strongly seropositive for antibodies to E2/NS1 protein of HCV was
included in the reaction mixture with each of the monoclonal antibodies at
a final concentration of 10%. Microtiter EIA was carried out as described
in Example 1. A greater than 50% inhibition in the binding of the
monoclonal antibody to the respective protein by the immune human sera was
considered as competitive (data presented in Table 1). Monoclonal
antibodies H13C113 and H23C163 were not significantly competed by sera
from individuals seropositive for antibodies to HCV E2/NS1.
Isotype
The isotypes of each of the monoclonal antibodies was determined by using
an isotyping kit (Amersham, Arlington Heights, Ill.) and following the
instructions included with it. Briefly, the tissue culture supernatant of
each monoclonal antibody and appropriate controls were reacted at a 1:5
dilution with strips coated with specific anti-isotype antibody, provided
in the kit described above. Assay protocol was followed exactly according
to the manufacturer's instructions. The isotype of each monoclonal
antibody of the invention is provided in TABLE 1.
EXAMPLE 6
Epitope Mapping
Monoclonal antibodies generated against the synthetic peptide (SEQ. ID. NO.
6) were mapped to the specific region of the HCV E2/NS1 protein by (a)
Western blot reactivity of each of the monoclonal antibodies with
subfragments of the HCV E2/NS1 protein and (b) reactivity with several
synthetic peptides selected for respective protein sequences, by
microtiter EIA using the procedure described in Example 1.
Reactivity of Monoclonals to Various Subfragments of Recombinant HCV NS1
proteins
Briefly, several individual oligonucleotides representing a.a. 365-731 of
HCV genome were ligated and cloned as three separate EcoRI-BAMHI
subfragments into the CKS fusion vector pJ0200. These three subfragments
were designated as pHCV80 (a.a. 365-731) (SEQ. ID. NO. 7), pHCV77 (a.a.
365-579) (SEQ. ID. NO. 8), and pHCV65 (a.a. 565-731) (SEQ. ID. NO. 9), as
illustrated in FIG. 2. The detailed methods for cloning and expression of
the CKS-fusion proteins are as disclosed in U.S. patent application Ser.
No. 07/610,180 and 07/572,822, which enjoy common ownership and are
incorporated herein by reference. Cell lysates of these clones were used
as antigens on Western blot analysis using the protocol described in
Example 5 for preliminary epitope mapping of anti-NS1 monoclonal
antibodies. FIG. 2 shows the binding of monoclonal antibodies H13C113 and
H23C163 to recombinant HCV E2/NS1 protein subfragments, wherein lane1
(normal human sera), lane 2(HCV immune human sera), and lane 3(normal
mouse sera) were included as controls. Lane 4 contains hybrid supernatant
from which H13C113 was cloned, lane 6 contains monoclonal antibody
H13C113, lane 5 contains a sister clone of monoclonal antibody H13C113
(H13C44), lane 10 contains monoclonal antibody H23C163, while lanes 8 and
9 contain sister clones of monoclonal antibody H23C163 (H23C41 and H23C41
respectively). Data for epitope mapping with these subfragments are
illustrated in FIG. 2. Monoclonal antibodies H13C113 and H23C163 showed
reactivity with pHCV 80 (SEQ. ID. NO. 7) and pHCV 65 (SEQ. ID. NO. 9)
which indicated the reactivity with HCV a.a. 564-731 (SEQ. ID. NO. 9).
Reactivity with Synthetic Peptides
Several amino acid sequences were selected from different regions of HCV
protein NS1 based on the PEPSCAN analysis as described in Example 1. A
list of the peptides used for the epitope mapping of these monoclonal
antibodies is listed below in TABLE 2.
TABLE 2
__________________________________________________________________________
Epitope Mapping with Synthetic Peptides
REGION OF
MONOCLONAL REACTIVITY OF
HCV GENOME
TESTED PEPTIDE a.a.
EACH WITH PEPTIDE
__________________________________________________________________________
NS1 H13C113 sp 643-663
sp 643-663
H23C163 sp 643-663
sp. 643-683
sp. 666-683
__________________________________________________________________________
Each of these peptides were assembled on a resin support by a stepwise
solid phase synthesis, starting with the carboxy terminal residue. A
procedure was employed similar to that described in E. Gross and T.
Heinehofer, eds., Barary and Merrifield, The Peptides 2:1284, Academic
Press, New York, N.Y. (1980), using a reaction vessel of an Applied
Biosystems Synthesizer Model 430A. After cleavage of the peptide from the
resin, the peptide was washed with diethyl ether and extracted in 40%
acetic acid solution. Crude peptide obtained after lyophilization of the
aqueous solution was employed as the antigen target for epitope mapping
experiments. Briefly, each of the peptides tested was coated on microtiter
wells at a concentration of 10 .mu.g/ml in bicarbonate buffer at pH 9.5.
EIA was performed in the manner described in Example 1. Monoclonal
antibody showing reactivity four times the negative control was considered
positive.
In addition, monoclonal antibodies to HCV NS1 were also mapped with PEPSCAN
analysis as described in Example 1. An EIA was performed with each of the
monoclonal antibodies to HCV NS1 by the procedures similar to one outlined
in Example 1 using the tissue culture supernatants of monoclonal
antibodies as the primary antibody and goat anti-mouse HRPO as the
secondary antibody with overlapping hexamer peptides encompassing a.a.
600-720 (SEQ. ID. NO. 1) of the HCV genome. Data are illustrated in FIG. 3
and FIG. 4. Monoclonal antibody H13C113 and H23C163 specifically reacted
with peptide sequence GDRCDLE (a.a. 649-655) (SEQ. ID. NO. 10) of the HCV
genome.
EXAMPLE 7
EIA for the Detection of HCV Proteins in Biological Samples Preparation of
Rabbit Polyclonal Antibodies Against HCV E2/NS1 Region
Young rabbits (3-4 months old and weighing approximately 2-3 kg) (available
from Hazelton LAbs, Denver Pa.) are immunized with 100-150 .mu.g of highly
purified HCV E2/NS1 synthetic peptide or the E2/NS1 recombinant proteins
cloned and expressed in either eukaryotic or prokaryotic systems as
described in Example 1 in Freund's complete adjuvant by intra-muscular
(i.m.) injection at four different sites. Subsequently, two immunizations
are carried out at two week intervals in similar fashion in Freund's
incomplete adjuvant. Immune response of the rabbits is monitored by EIA
and Western blot analysis. Rabbits are bled when acceptable immune
response to the protein is achieved. IgG from the immune rabbit sera is
purified by Protein-A sepharose affinity chromatography, by methods known
to those in the art.
Coating of Solid Phase
Rabbit IgG is prepared as herein described and then is coated on
polystyrene beads as the solid support for capture of E2/NS1 antigens in
test samples. The polystyrene beads are washed with distilled water and
incubated at 40.degree. C. for two hours with 5-10 .mu.g/ml of purified
HCV E2/NS1 synthetic peptide rabbit IgG in a buffer solution (0.1M Tris,
0.5M NaCl, 0.0022% Triton X-100.RTM., pH 8.5). The beads are washed once
with PBS and then soaked in 0.1% Triton X-100.RTM. in PBS for
approximately one hour at 40.degree. C. After washing twice with PBS, the
beads are overcoated with 3% bovine serum albumin (BSA) in PBS for
approximately one hour at 40.degree. C. Finally, the beads are overcoated
with 5% sucrose solution in PVS and dried under nitrogen. Anti-HCV human
polyclonal IgG, purified from sera of individuals seropositive for HCV
antibodies to E2/NS1 also is coated in similar fashion.
EIA
Monoclonal antibodies specific for HCV E2/NS1 are screened for use as the
probe for detection of HCV proteins in a test sample by EIA. Briefly, each
of the monoclonal antibodies is incubated with the E2/NS1 antigen in the
presence of polystyrene beads coated with anti-HCV rabbit polyclonal IgG.
The protocol for EIA is similar to that described hereinbelow.
200 .mu.l of test specimen suspected of containing antigen to HCV E2/NS1
protein is incubated in a reaction tray with 50 .mu.l of monoclonal
antibody of the invention (at a final protein concentration of about 5-10
.mu.g/ml diluted in a buffer containing 20 mM Tris, 0.1 mM NaCl, 1 mM
EDTA, 3.0% BSA, 0.3% Tween-20.RTM. and 10% FBS at pH 7.5), and a bead
coated with HCV rabbit IgG (prepared as described hereinabove). Overnight
incubation at ambient room temperature is performed, and then the beads
are washed with distilled water and 200 .mu.l of appropriately diluted
Horseradish Peroxidase labeled goat anti-mouse IgG (H & L) (Jackson
Immunoresearch, West Grove, Pa.) is added. Incubation with the labeled
probe is carried out at about 40.degree. C. for approximately two hours.
Beads are washed and transferred to reaction tubes containing 300 .mu.l of
O-phenylenediamine (OPD) color reagent. The reaction is carried out at
ambient room temperature in the dark for about 30 minutes, and then it is
stopped by the addition of 1 ml of 1N H.sub.2 SO.sub.4. Absorbance is
recorded at 492/600 nm. A negative control previously screened and
confirmed to be negative for NANBH infection is included in the
experiment. The positive control consists of a solution of synthetic
peptide to E2/NS1 in the buffer solution described hereinabove.
Triplicates of both positive and negative control are included with each
set of experiments.
In order to determine the efficiency of the antigen capture assay for the
detection of HCV E2/NS1 in a sample, various concentrations of recombinant
HCV E2NS1 synthetic peptide, ranging from 100 ng peptide/ml to 100 pg
peptide/ml are diluted in the buffer mentioned hereinabove. The EIA
procedure described above is performed with each of the diluted panel
members. For the purposes of comparison, each of the panel members is
tested with (a) anti-HCV rabbit polyclonal antibody on the solid phase and
(b) anti-HCV human polyclonal antibody on the solid phase. The efficiency
of the assay then is determined by evaluating data obtained.
The hybridomas which produce the monoclonal antibodies of the invention are
identified as hybridoma H13C113 producing monoclonal antibody H13C113, and
hybridoma H23C163 producing monoclonal antibody H23C163. Hybridomas
H13C113 and H23C163 were deposited at the American Type Culture Collection
(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 as of Aug. 20, 1991,
and have been accorded the following deposit numbers: hybridoma H13C113
was accorded ATCC deposit number HB 10857, and hybridoma H23C163 was
accorded ATCC deposit number HB 10856.
Thus, the novel monoclonal antibodies of the invention can be used in a
variety of ways. These monoclonal antibodies can be used for
immunoprecipitation of amplified product and detection of HCV nucleic acid
microparticles or carrier coated with anti-HCV monoclonal antibody used to
capture virus or viral protein associated with HCV RNA. Then detection
methodology for RNA may be used. An example of this type of assay is
taught in pending U.S. patent application Ser. No. 07/568,663, entitled A
METHOD FOR AMPLIFYING AND DETECTING A TARGET NUCLEIC ACID SEQUENCE, which
enjoys common ownership and is incorporated herein by reference.
These monoclonal antibodies also can be used for localization of HCV
antigens within the cell using HCV monoclonal antibody tagged directly
(fluorescence, colloidal gold, etc.) or using secondary tagged anti-mouse
antibody. Histopathology of disease may be tracked. Further, the detection
of native or recombinant HCV antigens in sera, tissue, cells, culture
media, or body fluid using individual monoclonal antibodies in a sandwich
configuration or a cocktail of monoclonal antibodies on the solid phase
and in the detection system.
One step antigen assays using monoclonal antibodies against non overlapping
epitopes may also be performed. Some monoclonal antibodies may recognize
antigenic epitopes not recognized by the infected individual and therefore
may be possible to recognize serum Ag both free and bound with human
antibody. Furthermore, "cryptic" or hidden antigens or antigenic
determinants may be uncovered by treatment of specimen with detergent or
reducing agent or both. For example, CORE antigen may exist in a capsid
form covered by the virus envelope. Stripping the envelope with detergent
should expose CORE antigen. Monoclonal antibodies may also offer pragmatic
advantages over high titer polyclonal antibody in giving greater
sensitivity in assay or allowing shorter incubation times.
Further, antibody immunoassays, one or two step competitive assays, were
developed in which anti-HCV competed with labeled anti-HCV monoclonal
antibody for binding to a limited number of antigenic sites. A more
sensitive competitive assay may be developed in which human anti-HCV binds
to HCV Ag in solution blocking or inhibiting the HCV Ag binding in HCV Ag
sandwich assay. Competitive assays using monoclonal antibodies allow a
more precise mapping of human antibody epitopes and may be useful for
determining virus neutralizing antibody epitopes. Some monoclonal
antibodies may have virus neutralizing activity. Finally, monoclonal
antibodies should be useful in immunoaffinity purification of native viral
and recombinant HCV antigens and proteins.
Other variations of applications of the use of these unique monoclonal
antibodies provided herein include the detection of HCV in immune
complexes, or latent and/or cryptic antigens, and/or associated with viral
nucleic acid for detection of the nucleic acid by PCR, LCR, or by direct
hybridization. Still other variations and modifications of the specific
embodiments of the invention as set forth herein will be apparent to those
skilled in the art. Accordingly, the invention is intended to be limited
only in accordance with the appended claims.
TABLE 1
__________________________________________________________________________
MONOCLONAL ANTIBODIES TO HCV NS1 PROTEIN
COMP
WITH
IMMUNE
WESTERN BLOT
TITER EPITOPE
IMMUNOGEN
MAB ID
ISOTYPE
HU.SERA
pHCV-65.sup.a
pHCV-80.sup.b
643-683
pHCV80
HCV A.A.
__________________________________________________________________________
sp 643-683
H13C113
IgG3,k
- + + 10 ng/ml
80 ng/ml
.sup. 649-655.sup.c
H23C163
IgG2b,k
- + + 80 ng/ml
1 ug/ml
649-655
__________________________________________________________________________
.sup.a pHCV-65 a.a. 565-731
.sup.b pHCV-80 a.a. 365-731
.sup.c a.a. sequence = Gly--Glu--Arg--Cys--Asp--Leu--Glu
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 10
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Gly ProTrpIleThrProArgCysLeuValAspTyrProTyrArgLeu
151015
TrpHisTyrProCysThrIleAsnTyrThrIlePheLysIleArgMet
202530
TyrValGlyGlyValGluHisArgLeuGluAlaAlaCysAsnTrpThr
354045
ArgGly GluArgCysAspLeuGluAspArgAspArgSerGluLeuSer
505560
ProLeuLeuLeuThrThrThrGlnTrpGlnValLeuProCysSerPhe
65 707580
ThrThrLeuProAlaLeuSerThrGlyLeuIleHisLeuHisGlnAsn
859095
IleVal AspValGlnTyrLeuTyrGlyValGlySerSerIleAlaSer
100105110
TrpAlaIleLysTrpGluTyrValVal
115120
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CysLeuValAspTyrProTyrArgLeuTrpHisTyrProCysThrIle
151015
AsnTyrThrIlePhe
20
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AlaCysAsnTrpThrArgGlyGluArgCysAspLeuGluAspArgAsp
151015
ArgSerGluLeuSer
20
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
LeuLeuThrThrThrGlnTrpGlnValLeuProCys SerPheThrThr
151015
LeuPro
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GlnTrpGlnValLeuProCysSerPheThrThrLeuProAlaLeuSer
151015
ThrGlyLeuIleHis
20
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AlaCysAsnTrpThrArgGlyGluArgCysAspLeuGluAspArg Asp
151015
ArgSerGluLeuSerProLeuLeuLeuThrThrThrGlnTrpGlnVal
202530
LeuProCysSerPheThrThrLeuPro
3540
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 621 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:7:
MetSerPheValValIleIleProAlaArgTyrAlaSerThrArgLeu
151015
ProGlyLysProLeuValAspIleAsnGlyLysProMe tIleValHis
202530
ValLeuGluArgAlaArgGluSerGlyAlaGluArgIleIleValAla
3540 45
ThrAspHisGluAspValAlaArgAlaValGluAlaAlaGlyGlyGlu
505560
ValCysMetThrArgAlaAspHisGlnSerGlyThrGluArgLeuAla
65707580
GluValValGluLysCysAlaPheSerAspAspThrValIleValAsn
8590 95
ValGlnGlyAspGluProMetIleProAlaThrIleIleArgGlnVal
100105110
AlaAspAsnLeuAlaGlnArgGlnValGlyMetThrThrLeuA laVal
115120125
ProIleHisAsnAlaGluGluAlaPheAsnProAsnAlaValLysVal
130135140
Val LeuAspAlaGluGlyTyrAlaLeuTyrPheSerArgAlaThrIle
145150155160
ProTrpAspArgAspArgPheAlaGluGlyLeuGluThrValGlyAs p
165170175
AsnPheLeuArgHisLeuGlyIleTyrGlyTyrArgAlaGlyPheIle
180185190
ArgArgTyrValAsnTrpGlnProSerProLeuGluHisIleGluMet
195200205
LeuGluGlnLeuArgValLeuTrpTyrGlyGluLysIleHisValAla
210215220
ValAlaGlnGluValProGlyThrGlyValAspThrProGluAspLeu
225230235240
AspProSerThrAsnSerThrMetValGlyAsnTrpAlaLysValLeu
245250255
ValValLeuLeuLeuPheAlaGlyValAspAlaGluThrHisVal Thr
260265270
GlyGlySerAlaGlyHisThrValSerGlyPheValSerLeuLeuAla
275280285
ProGlyAlaLysGlnAsnValGlnLeuIleAsnThrAsnGlySerTrp
290295300
HisLeuAsnSerThrAlaLeuAsnCysAsnAspSerLeuAsnThrGly
305 310315320
TrpLeuAlaGlyLeuPheTyrHisHisLysPheAsnSerSerGlyCys
325330335
ProGluArgLeuAlaSerCysArgProLeuThrAspPheAspGlnGly
340345350
TrpGlyGlnIleSerTyrAlaAsnGlySerGlyProAspGlnArgPr o
355360365
TyrCysTrpHisTyrProProLysProCysGlyIleValProAlaLys
370375380
SerVal CysGlyProValTyrCysPheThrProSerProValValVal
385390395400
GlyThrThrAspArgSerGlyAlaProThrTyrSerTrpGlyGluAsn
405410415
AspThrAspValPheValLeuAsnAsnThrArgProProLeuGlyAsn
420425430
TrpPheGlyCysThrTrpMetAsnSerThrGlyPheThrLysValCys
435440445
GlyAlaProProCysValIleGlyProProCysValIleGlyGlyAla
450455460
GlyAsnAsnThrLeuHisCysProThrAspCysPheArgLysHisPro
465470475480
As pAlaThrTyrSerArgCysGlySerGlyProTrpIleThrProArg
485490495
CysLeuValAspTyrProTyrArgLeuTrpHisTyrProCysThrIle
500505510
AsnTyrThrIlePheLysIleArgMetTyrValGlyGlyValGluHis
515520525
Arg LeuGluAlaAlaCysAsnTrpThrArgGlyGluArgCysAspLeu
530535540
GluAspArgAspArgSerGluLeuSerProLeuLeuLeuThrThrThr
545 550555560
GlnTrpGlnValLeuProCysSerPheThrThrLeuProAlaLeuSer
565570575
ThrGlyLeuIleHisLeuHisGlnAsnIleValAspValGlnTyrLeu
580585590
TyrGlyValGlySerSerIleAlaSerTrpAlaIleLysTrpGluTyr
595600605
ValValLeuLeuPheLeuLeuLeuAlaAspAlaArgVal
610615620
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 414 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
MetSerPheValValIleIleProAlaArgTyrAlaSerThrArgLeu
15 1015
ProGlyLysProLeuValAspIleAsnGlyLysProMetIleValHis
202530
ValLeuGluArgAlaArgGluSer GlyAlaGluArgIleIleValAla
354045
ThrAspHisGluAspValAlaArgAlaValGluAlaAlaGlyGlyGlu
5055 60
ValCysMetThrArgAlaAspHisGlnSerGlyThrGluArgLeuAla
65707580
GluValValGluLysCysAlaPheSerAsp AspThrValIleValAsn
859095
ValGlnGlyAspGluProMetIleProAlaThrIleIleArgGlnVal
1001 05110
AlaAspAsnLeuAlaGlnArgGlnValGlyMetThrThrLeuAlaVal
115120125
ProIleHisAsnAlaGluGluAlaPheAsnP roAsnAlaValLysVal
130135140
ValLeuAspAlaGluGlyTyrAlaLeuTyrPheSerArgAlaThrIle
145150155 160
ProTrpAspArgAspArgPheAlaGluGlyLeuGluThrValGlyAsp
165170175
AsnPheLeuArgHisLeuGlyIleTyrGl yTyrArgAlaGlyPheIle
180185190
ArgArgTyrValAsnTrpGlnProSerProLeuGluHisIleGluMet
195200 205
LeuGluGlnLeuArgValLeuTrpTyrGlyGluLysIleHisValAla
210215220
ValAlaGlnGluValProGlyThrGlyValAspThrPro GluAspLeu
225230235240
AspProSerThrAsnSerMetGlyAlaProProCysValIleGlyGly
245250 255
AlaGlyAsnAsnThrLeuHisCysProThrAspCysPheArgLysHis
260265270
ProAspAlaThrTyrSerArgCysGlySer GlyProTrpIleThrPro
275280285
ArgCysLeuValAspTyrProTyrArgLeuTrpHisTyrProCysThr
290295 300
IleAsnTyrThrIlePheLysIleArgMetTyrValGlyGlyValGlu
305310315320
HisArgLeuGluAlaAlaCysAsnTrpThrArgG lyGluArgCysAsp
325330335
LeuGluAspArgAspArgSerGluLeuSerProLeuLeuLeuThrThr
340345 350
ThrGlnTrpGlnValLeuProCysSerPheThrThrLeuProAlaLeu
355360365
SerThrGlyLeuIleHisLeuHisGlnAsnIleVa lAspValGlnTyr
370375380
LeuTyrGlyValGlySerSerIleAlaSerTrpAlaIleLysTrpGlu
385390395 400
TyrValValLeuLeuPheLeuLeuLeuAlaAspAlaArgVal
405410
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 463 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
MetSerPheValValIleIleProAlaArgTyrAlaSerThrArgLeu
151015
ProGlyL ysProLeuValAspIleAsnGlyLysProMetIleValHis
202530
ValLeuGluArgAlaArgGluSerGlyAlaGluArgIleIleValAla
354045
ThrAspHisGluAspValAlaArgAlaValGluAlaAlaGlyGlyGlu
505560
ValCysMetThrArgAlaA spHisGlnSerGlyThrGluArgLeuAla
65707580
GluValValGluLysCysAlaPheSerAspAspThrValIleValAsn
859095
ValGlnGlyAspGluProMetIleProAlaThrIleIleArgGlnVal
100105110
AlaAspAsnLeu AlaGlnArgGlnValGlyMetThrThrLeuAlaVal
115120125
ProIleHisAsnAlaGluGluAlaPheAsnProAsnAlaValLysVal
130 135140
ValLeuAspAlaGluGlyTyrAlaLeuTyrPheSerArgAlaThrIle
145150155160
ProTrpAspArgAspA rgPheAlaGluGlyLeuGluThrValGlyAsp
165170175
AsnPheLeuArgHisLeuGlyIleTyrGlyTyrArgAlaGlyPheIle
18 0185190
ArgArgTyrValAsnTrpGlnProSerProLeuGluHisIleGluMet
195200205
LeuGluGlnLeuArgVa lLeuTrpTyrGlyGluLysIleHisValAla
210215220
ValAlaGlnGluValProGlyThrGlyValAspThrProGluAspLeu
225230 235240
AspProSerThrAsnSerThrMetValGlyAsnTrpAlaLysValLeu
245250255
ValValLeuLeuLeu PheAlaGlyValAspAlaGluThrHisValThr
260265270
GlyGlySerAlaGlyHisThrValSerGlyPheValSerLeuLeuAla
275 280285
ProGlyAlaLysGlnAsnValGlnLeuIleAsnThrAsnGlySerTrp
290295300
HisLeuAsnSerThrAlaLeuAsn CysAsnAspSerLeuAsnThrGly
305310315320
TrpLeuAlaGlyLeuPheTyrHisHisLysPheAsnSerSerGlyCys
325 330335
ProGluArgLeuAlaSerCysArgProLeuThrAspPheAspGlnGly
340345350
TrpGlyGlnIleSerT yrAlaAsnGlySerGlyProAspGlnArgPro
355360365
TyrCysTrpHisTyrProProLysProCysGlyIleValProAlaLys
370 375380
SerValCysGlyProValTyrCysPheThrProSerProValValVal
385390395400
GlyThrThrAspArgSerGl yAlaProThrTyrSerTrpGlyGluAsn
405410415
AspThrAspValPheValLeuAsnAsnThrArgProProLeuGlyAsn
420 425430
TrpPheGlyCysThrTrpMetAsnSerThrGlyPheThrLysValCys
435440445
GlyAlaProProCysValIle GlyGlyAlaGlyAsnAsnThrLeu
450455460
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GlyAspArgCysAspLeuGlu
15
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